HCV is the major causative agent for post-transfusion and for sporadic non A, non B hepatitis (Alter, H. J. (1990) J. Gastro. Hepatol. 1:78-94; Dienstag, J. L. (1983) Gastro 85:439-462). Despite improved screening, HCV still accounts for at least 25% of the acute viral hepatitis in many countries (Alter, H. J. (1990) supra; Dienstag, J. L. (1983) supra; Alter, M. J. et al. (1990a) J.A.M.A. 264:2231-2235; Alter, M. J. et al (1992) N. Engl. J. Med. 327:1899-1905; Alter, M. J. et al. (1990b) N. Engl. J. Med. 321:1494-1500). Infection by HCV is insidious in a high proportion of chronically infected (and infectious) carriers who may not experience clinical symptoms for many years. The high rate of progression of acute infection to chronic infection (70-100%) and liver disease (&gt;50%), its world-wide distribution and lack of a vaccine make HCV a significant cause of morbidity and mortality.
HCV is an enveloped virus whose genome is a 9.4 kb single-stranded RNA (sense(+)) encoding a single polyprotein that is processed by proteolysis to yield at least 9 proteins. HCV is related to pestiviruses and flaviviruses (Choo, Q-L. et al. (1989) Science 244:362-364; Choo, Q-L. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455. Reinfection of previously HCV-infected chimpanzees suggests that protective immunity is transient or non-existent (Farci, P. et al (1992) Science 258:135-140). Furthermore, results of recent vaccine trials suggest that development of an effective vaccine is remote (Houghton, M. et al. (1994) 2nd Internat. Meeting on Hepatitis C (San Diego)). Attempted treatment of chronic HCV infection using existing antiviral agents produces low cure rates and serious side effects. (Dienstag, J. L. (1983) supra.)
The nucleotide sequence of the HCV genome has been cloned and a single open reading frame has been identified. Using a vaccinia virus expression system, several cleavage products have been tentatively identified. (Lin, C. et al. (1994) J. Virol. 68:5063-5073; Grakoui, A. et al. (1993) J. Virol. 67:1385-1395.) The various putative cleavage products were recognized by antibodies raised against various peptides synthesized from amino acid sequences deduced from various segments of the coding regions. Sizes of antibody-reactive peptides were estimated by SDS-PAGE (See FIG. 1). The non-structural protein designated 5B (NS5B) has been shown to have an amino-terminal sequence SMSY (Ser-Met-Ser-Tyr). The NS5B region encodes a 68 kd protein (p68) which contains an internal GDD (Gly-Asp-Asp) motif found in RNA-dependent RNA polymerases of other RNA viruses (Koonin, E. V. (1991) J. Gen. Virol. 72:2197-2206). However, no polymerase activity has been detected for HCV p68. In fact, the question has been raised that the 5B protein (p68) alone does not encode an active RNA-dependent RNA polymerase enzyme and that another subunit, possibly the NS5A gene product, is essential to catalytic activity. Prior attempts by the inventors and others to express the NS5B coding region as a fusion protein, using existing expression systems that facilitate purification of the fusion product and specific cleavage have failed to yield any active polymerase.